We have examined hybrid viral RNAs synthesized in both human and monkey cells infected by three nondefective adenovirus-SV40 hybrid viruses. Most of the hybrid viral RNA molecules appear to initiate within adenoviral sequences, but are polyadenylated on their 3' end at the early SV40 mRNA polyadenylation site. The Ad2 ion ND4 stock of virus is not homogenous, but consists of two principal populations of viral DNA. Both populations contain a segment of SV40 DNA extending from SV40 map positions 0.63 to 0.11 in a left to right orientation at adenovirus map position 0.79. One population contains an intack SV40 segment while the other (representing 80 to 85% of the population) has a 500 base-pair deletion mapping from approximately 0.60 to 0.50 SV40 map units. This deletion encompasses the SV40 DNA segment which encodes the early SV40 splice sites. Cells infected by the mixed Ad2 ion ND4 population induce the synthesis of at least three major SV40-containing RNA molecules among the hybrid viral transcripts. The most abundant of these hybrid mRNAs appears only late in the lytic cycle, after the onset of viral DNA replication.